The Study About Effects Of Electro-acupuncture Therapy On The Regional Cerebral Blood Flow And Angiogenesis Of The Cerebral Ischemia Reperfusion Rats Cortex

Objective Ischemic cerebrovascular disease(ICVD), with highmorbidity, mortality and recurrence rate, has become one of thethree killers to threaten human being health. Nowadays, the mostquantity of research about Ischemic cerebrovascular disease is therelationship between the regional cerebral blood flow (rCBF) andangiogenesis after cerebral ischemia reperfusion. Although bloodflow rescues the nerve cells after ischemia reperfusion, it willincrease nerve cells damage and even lead to their death. The r-estoration of nerve cells after ischemic cerebrovascular diseaseis not only associated with the expression of cell factor and oxygensupply, but also depends more on the degree of ischemic angiogenesis.As angiogenesis can affect the establishment of collateral circ-ulation after cerebral ischemia reperfusion, and create a goodenvironment for the reshaping of the nerve cells and nerve repairfunction. Microvascular endothelial cells are the basic unit of thecomposition of blood vessels. The main manifestation of angioge-nesis is the change of the microvascular endothelial cells, inclu-ding the change in form and the increased number of them. The pr-oliferation of vascular endothelial cells and the increase of dens- ity of the newborn blood vessels all provide the basis for thereperfusion of blood flow to ischemic brain, and the regionalcerebral blood flow is the most intuitionistic index to evaluatematurity of new born blood vessels. Because electro acupuncturehas a protective effect on reducing ischemic brain damage in mu-ltiple levels of targets and different ways, observing the influ-ence of the relationship between the regional cerebral blood flowand angiogenesis of ischemia brain after acupuncture could furtherstudy the mechanism in order to grasp opportunity of ac-upunctureand moxibustion as a treatment on cerebral ischemia exactly. Now,Erythropoietin(EPO) is the most popular cytokines on promotingangiogenesis，because it can go through the blood brain barrierrapidly without increasing its permeability, inhibit the inflam-matory reaction, and protect the neurons, at the same time, EPOcan also protect the ischemic brain tissue by activating the do-wnstream JAK2/STAT5molecular signaling pathways and can affectthe Neuro vascular unit (NVU).But after cerebral ischemia reper-cussion, with its time prolonging, EPO gene and protein from is-chemic brain tissue would change, the electro acupuncture couldinfluence the expression of EPO in different degrees, differenttime and different ischemic areas. The expression of cytokines canpromote JAK protein and STAT protein phosphorylation after cere-bral ischemia reperfusion. Electro-acupuncture can inhibit infla-mmation, induce apoptosis and reduce brain damage by emulating andcontrolling the JAK1/STAT3molecular signaling pathways. A latestresearch, JAK2/STAT5which is a molecular signaling pathway isrelated to angiogenesis, so whether angiogenesis of cerebral is-chemia after electro-acupuncture is related to activation of the JAK2/STAT5signaling pathways is one of the purposes of this study.By observing the local cerebral blood flow changes from the cortexof the rats that were made focal cerebral ischemia repercussion,the impact of microvascular endothelial cells and the expressionof EPO protein and EPO mRNA in cerebral cortex and the changes inexpression of P-JAK2,P-STAT5that are related to the molecularsignaling pathways downstream, we can explore the possible opp-ortunity, targets and mechanisms about the treatment to ischemiccerebrovascular disease by using electro-acupuncture.Methods100Sprague-Dawley rats with clean grade were randomlydivided into a control group (n=10),a sham-operation group (n=10),a model group(n=40) and a electro-acupuncture group(n=40). Acco-rding to the time of cerebral ischemia reperfusion twelve hours,twenty-four hours, forty-eight hours and seventy-two hours, thetwo later groups were further divided into four subgroups respe-ctively with10cases in each group. The model of focal cerebralischemia reperfusion was established by inserting nylon into themiddle cerebral artery. After forty-five minutes of the middlecerebral artery occlusion (MCAO),the blood flow was restored ex-cept the control group and the sham-operation group which was onlyseparated the Internal carotid artery and the External carotidartery from the Common carotid artery. The rats in the model groupand the electro-acupuncture group were made focal cerebral isch-emia repercussion model based on the method above. According tothe five levels of Zea-Longa criteria to score of experimentalanimal neurological disorder, only nerve dysfunction in more thanone level of rats can be retained as building success. The ratsof electro-acupuncture group were treated with electro-acupun- cture at Quchi(LI11)and Zusanli(ST36) with density wave (frequ-ency:2Hz/100Hz,intensity:1Am)for thirty minutes and other groupswithout any treatment. This treatment was repeated every24hours.By using the microscope to observe the pathological changes of ratsbrain cortex after cerebral ischemia reperfusion, which has beenstained by Eosin Staining Solution. At the same time, the regionalcerebral blood flow was measured using a Doppler laser blood streamdetector before cerebral ischemia reperfusion and at differenttimes(12h,24h,48h and72h) after cerebral ischemia reperfusion.Immunofluorescence technique detected the numbers of microvasc-ular endothelial cells of local cerebral ischemic cortex. Theexpression of EPO protein of local cerebral ischemia cortex indifferent times was detected by Immunohistochemistry; the levelof EPO mRNA in cortex of rats at different time (12h,24h,48h and72h) after cerebral ischemia reperfusion was determined by realtime Polymerase Chain Reaction. The content of the P-JAK2andP-STAT5protein in cortex of rats at different times(12h,24h,48hand72h) after cerebral ischemia reperfusion was measured byWestern Blotting.Results (1) By using the microscope to observe the pathologicalchanges of rats brain cortex after cerebral ischemia reperfusion,we could find the brain cortex of the rats from the control groupwas regular, which dyeing was uniform, and the corticocerebralstructure was clear and compact. But brain cortex of the sham-operation group had only very small amounts of inflammatory cells.After cerebral ischemia reperfusion, the rates cortex appeareddifferent degree of damage, which organizational structure wasdisorder and the corticocerebral interstitial was edema. In the meantime, the gap around microvascular became widening, micrangiumdeformation and the part of micrangium expansion had happen. Eventhe deformation and necrosis of neurons of rats ischemia cortex andthe exudation of inflammatory cells and glial cells were detected.But the injury of ischemic brain cortex of the rats from electro-acupuncture group was a significant alleviated that of the modelgroup. That was because electro-acupuncture could relieved injuryof the ischemic cerebral cortex of rats after cerebral ischemiarepercussion effectively. Meanwhile it could corrected the diso-rdered structure of the damaged brain tissue and the degree ofswelling, it also could narrowed the ischemia area, and even reducedthe number of dead cells and the degeneration degree of it, moreoverit could protected the micrangium in ischemic region. The regionalcerebral blood flow was measured by using the Doppler laser bloodstream detector, which from the control group and the sham-oper-ation group was no change before and after the experiment. Howeverthe regional cerebral blood flow of the model group and electro-acupuncture group reduced in different level. As the change ofcerebral ischemia reperfusion time, the regional cerebral bloodflow of above two groups appeared upward tendency. Electro-acu-puncture could increase regional cerebral blood flow after ischemiareperfusion obviously. The regional cerebral blood flow of elec-tro-acupuncture group was significantly more than that of the modelgroup in different times except at24h after cerebral ischemiareperfusion (P<0.05),and the phenomenon was more obviously at72hours’reperfusion（p<0.01).(2)By using the fluorescence micr-oscope to observe the numbers of microvascular endothelial cellsof local cerebral ischemic cortex, there was no change of numbers of microvascular endothelial cells of the control group before andafter the experiment. And local cerebral ischemic cortex of the ratsin the sham-operation group appeared proliferation of a handfulmicrovascular endothelial cells, and the comparison of the micr-ovascular endothelial cells proliferation between the controlgroup and the sham-operation group was no statistical difference（P>0.05).After cerebral ischemia reperfusion, the microvascularendothelial cells from cerebral cortex appeared a certain degreeof proliferation. With ischemia reperfusion time prolonging, theproliferation of microvascular endothelial cells began increasingfrom the ischemia reperfusion12th hour, keeping rising during thefollowing ischemia reperfusion12h, reaching the top of quantityat ischemia reperfusion48th hour, and at last decreased at ischemiareperfusion72th hour. The number of microvascular endothelialcells in electro acupuncture group was significantly more than thatin the model group all the time（P<0.05，P<0.01）,except12hour’ischemia reperfusion（P>0.05).The regional cortical blood flow andthe number of microvascular endothelial cell proliferation werestatistically analyzed by Pearson Correlation statistical analysis.The result of that was a positive correlation between the regionalcortical blood flow and the number of microvascular endothelialcell proliferation in model group and electro-acupuncture group.This means that with the ischemia reperfusion time changing, thechanging trend of the number of microvascular endothelial cellproliferation would keep pace with the regional cortical blood flowafter cerebral ischemia reperfusion.(3)To observing the expressionof EPO protein and the level of the EPO mRNA were in different levelsby using microscope. There was no change of the expression of EPO protein of the control group before and after the experiment. Andlocal cerebral ischemic cortex of the rats in the sham-operationgroup expressed handful EPO protein, and the compared of the EPOprotein between the control group and the sh-am-operation groupwas no statistical difference（P>0.05).After cerebral ischemiareperfusion, with ischemia reperfusion time p-rolonging, theexpression of EPO protein and the level of EPO mRNA began increasingfrom the ischemia reperfusion12th hour, reaching the top of quan-tity in ischemia reperfusion24th hour, and they began decreasingat ischemia reperfusion48th hour and this trends would last tillischemia reperfusion72th hour. In comparison with model group, theprotein level of EPO in electro-acupuncture group was signifi-cantly more than that in the model group all the time except12hours’ischemia reperfusion（P>0.05).According to the result of the realtime Polymerase Chain Reaction, there was no change of the levelof EPO mRNA of the control group before and after the experiment.And local cerebral ischemic cortex of the rats in the sham-operationgroup expressed handful EPO mRNA, and the com-parison of the levelof the EPO mRNA between the control group and the sham-operationgroup was no statistical difference（P>0.05).The expression of EPOmRNA in electro-acupuncture group was always si-gnificantly morethan that in the model group（P<0.05、P<0.01).(4)On the basis ofthe result of the Western-Blotting protein im-printing, the con-tent of P-JAK2protein and the P-STAT5protein were in differentlevels. There was no change of the content of P-JAK2protein andP-STAT5protein in the control group before and after the experiment.The local cerebral ischemic cortex of the rats in the sham-operationgroup expressed handful the P-JAK2protein and P-STAT5protein, and the comparison of the two indexes between the control group and thesham-operation group was no statistical di-fference（P>0.05).Aftercerebral ischemia reperfusion, with ischemia reperfusion timeprolonging, these two indexes began increasing from the ischemiareperfusion12th hour, keeping rising during the following ischemiareperfusion12h, reaching the top of quantity at ischemia reperf-usion48th hour, and at last decreased at ischemia reperfusion72thhour. The P-JAK2protein content of electro-acupuncture group wasmore than that of model group in ischemia reperfusion12h andischemia reperfusion72h（P<0.05）.But, there is no difference aboutthe P-JAK2protein content from cerebral ischemic cortex of towgroup rats（P>0.05）at ischemia reperfusion12th hour and ischemiareperfusion48th hour. The protein content of P-STAT5of electro-acupuncture group was significantly more than that in the modelgroup（P<0.01）.Conclusion Electro-acupuncture can promote the regional cere-bral blood flow effectively, and increase the quantity of endothel-ial cells to promote angiogenesis. It can increase the expressionof EPO protein and EPO mRNA of the local cerebral ischemic cortexof the rats after cerebral ischemia reperfusion. Moreover, electro-acupuncture also can promote JAK2protein and STAT5protein phos-phorylation after cerebral ischemia reperfusion, and increase thecontent of P-JAK2protein and P-STAT5protein in JAK2/STAT5signaling pathways. All of these effects from electro-acupuncturecan perfect the pathological morphological changes and provideeffective cerebral protective to the ischemic brain tissue.